With annual global energy consumption predicted to double by 2050 and CO2 emissions having already reached crisis point, the growing demand for renewable and environmentally clean power sources has been propelled to the forefront of academic and industrial research. One strategy currently under investigation is the employment of enzymes in enzymatic fuel cells (EFCs), which convert chemical energy to electrical energy. This research focused on comparing the use of different protein-stabilising agents in low-cost bilirubin oxidase (BOD) from Myrothecium verrucaria cathodes with sandwich architectures, for biocatalysis of the electrochemical oxygen reduction reaction (ORR). As a novel route towards EFC cost reduction, Super P (carbon black powder, CBP) was initially selected as an electronically conductive enzyme support material. To increase direct electron transfer, COOH functionalisation of the CBP was investigated and characterised with ATR-FTIR, CHN and thermogravimetric analysis and the Boehm titration technique. Chemical oxidation yielded surface COOH concentrations ranging between 0.16 - 1.70 mmol g-1, which was determined with cyclic voltammetry (CV) to be outside of the suitable range for application within BOD cathodes. This resulted in the use of multi-walled carbon nanotubes (MWCNTs) as the enzyme support material. The effect of several room temperature ionic liquids (RTILs) on BOD activity was also investigated, resulting in the novel identification of three RTILs that are BOD-compatible in the absence of water: 1-ethyl-3 -methylimidazolium ethylsulfate (EMIM-EtSO4), 1-ethyl-3-methylimidazolium diethylphosphate (EMIM-Et2PO4) and 1,3-methylimidazolium dimethylphosphate (MMIM-Me2PO4). Two BOD cathodes, utilising the non-ionic surfactants (NISs) Triton X-100 and Tween 20, were developed, optimised and characterised. Triton X-100 was determined to be the favourable NIS for three-layer BOD cathode fabrication, compared to Tween 20, especially with respect to electrode current densities (-81 vs. -60 μA cm-2 at 0.3 V vs. Ag|AgCl at pH 5.0) attributed to the BOD-catalysed ORR. Chronoamperometric studies over a period of 8 days showed no difference between the stability of the two BOD cathodes